In most eukaryotic cells (including yeast and humans), the essential iron containing cofactors, heme and Fe-S clusters, are synthesized within mitochondria. Since the mitochondrial inner membrane must be impermeable to ions, a compartmentation problem is created - how does iron cross the mitochondrial membrane? We identified mutants of proteins of the mitochondrial carrier family that showed major and contrasting effects on iron distribution. Mrs3/mrs4 double mutants showed iron accumulation in the cytoplasm at the expense of mitochondria; Yhm1 mutants showed an opposite pattern, with iron accumulation in mitochondria at the expense of the cytoplasm. In our first aim, we will examine the effects of loss of function or overexpression of these mitochondrial carriers on iron import and export from mitochondria. Initial studies will be indirect, emphasizing effects on cellular iron uptake, iron partitioning and the status of iron proteins in different cellular compartments. Analysis of site directed mutants will correlate critical sequence motifs of the carrier proteins with the cellular phenotypes. In the second aim, a more direct assay for uptake of iron into mitochondria for heme synthesis has been developed and additional assays of iron transport (in and out) are being developed; these will be used to assess transport functions of these transporters in permeabilized cells in situ. The final aim will be to find other genes/proteins involved in transfer of iron from cytosol to mitochondria. A genome wide screen for mutations that lead to misregulated iron uptake will be undertaken. Synthetic lethal relationships between mrs3/4 or yhm1 and other genes may reveal new components of intracellular iron trafficking pathways. The organization of mitochondria is highly conserved with humans, and mitochondrial carrier proteins, including Mrs3/4 and Yhm1 have human orthologs. Therefore these studies will have implications for human diseases in which iron homeostasis plays a role, such as anemia and neurodegeneration.